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Hypothyroidism provides resistance to reperfusion injury following myocardium ischemia
The International Journal of Biochemistry & Cell Biology 33 (2001) 499–506 www.elsevier.com/locate/ijbcb
Hypothyroidism provides resistance to reperfusion injury following myocardium ischemia Israel Bobadilla a, Martha Franco b, David Cruz c, Jose` Zamora d, Sandra G. Robles a, Edmundo Cha´vez a,* Departamento de Bioquı´mica, Instituto Nacional de Cardiologı´a, Ignacio Cha´6ez, Juan Badiano c 1, Mexico, D.F. 014080, Mexico b Departamento de Nefrologı´a, Instituto Nacional de Cardiologı´a, Ignacio Cha´6ez, Mexico, D.F. 014080, Mexico c Departamento de Patologı´a, Instituto Nacional de Cardiologı´a, Ignacio Cha´6ez, Mexico, D.F. 014080, Mexico d Departamento de Endocrinologı`a, Instituto Nacional de Cardiologı´a, Ignacio Cha´6ez, Mexico, D.F. 014080, Mexico a
Received 16 May 2000; accepted 17 January 2001
Abstract A growing body of evidence has demonstrated that reperfusion injury may be mediated, in part, by mitochondrial Ca2 + overload that promotes non-selective permeability of the inner membrane. In this regard it is known that mitochondria from hypothyroid rats are resistant to membrane damage as induced by Ca2 + . The purpose of this study was to evaluate the sensitivity of hearts from hypothyroid rats, to the damage by reperfusion, after an ischemic period of 5 min. The results were compared with those from control and hyperthyroid rats. Hypothyroidism was established by surgical removal of the thyroid gland; in turn hyperthyroidism was induced after a daily injection of 2 mg/kg of 3,5,3%-triiodothyronine for 4 days. ECG tracings from hypothyroid rats showed a total absence of post-reperfusion arrhythmias conversely to what was observed in control and hyperthyroid rats. The release of creatine kinase and aspartate amino transferase to the plasma in hypothyroid rats was found to be lower than that found in hyperthyroid and euthyroid rats. The histological studies showed that myocardial fibers from hypothyroid rats were in good condition and retained their striae and a remarkable near absence of edema was clearly observed. © 2001 Published by Elsevier Science Ltd. Keywords: Hypothyroidism; Ischemia/reperfusion; Rat heart; Myocardium reperfusion; Reperfusion injury
1. Introduction Ischemia, when sufficiently prolonged, produces non-reversible tissue destruction. In the context of myocardial ischemia, advances in clinical and sur* Corresponding author. Tel.: + 52-5-5732911; fax: + 52-55730926. E-mail address: [email protected] (E. Cha´vez).
gical practice have allowed reperfusion of viable heart tissue, i.e. cardioplegic solutions, implant of intracoronary stents, or coronary by-pass. However, abrupt reoxygenation causes further substantial cell injury produced by oxygen-derived reactive species formed through dissimilar mechanisms [1–4]. In addition, the resulting cellular Ca2 + overload contributes significantly to the myocardial insult [5–7]. Regarding the latter, in a
1357-2725/01/$ - see front matter © 2001 Published by Elsevier Science Ltd. PII: S 1 3 5 7 - 2 7 2 5 ( 0 1 ) 0 0 0 1 6 - 4
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I. Bobadilla et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 499–506
previous work we demonstrated that the immunosuppressive drug cyclosporin A (CSA) avoids heart damage promoted by ischemia-reperfusion [8]. The ability of CSA to prevent mitochondrial membrane leakage, caused by Ca2 + overload is well documented [9– 11]. At present, there is a consensus about the central role of mitochondrial permeability transition in heart reperfusion injury [11,12]. Recently we found that hypothyroidism renders liver mitochondria resistant to permeability transition [13]. We proposed that the lack of cardiolipin in the lipid phase of membrane restrains the shift of adenine nucleotide translocase to a transmembrane pore. Taking together these previous published data we decided to explore the possibility that heart tissue of hypothyroid rats might be less sensitive to ischemia-reperfusion injury. The results indicate that, certainly, hypothyroidism conferred protection against reperfusion arrhythmias, and the release of creatine kinase (CK) and aspartate amino transferase (ASAT). Furthermore, histological studies show that myocardial tissue from hypothyroid rat hearts preserved its normal structure. When compared with experiments performed in hyperthyroid rats the picture was quite different. In these rats there was an increased incidence of ventricular tachycardia, an increase in the serum levels of CK and ASAT, as well as a generalized injury in the myocardial structure.
2. Materials and methods Hypothyroidism was induced in male Wistar rats, weighing between 250 and 300 g, by removing the thyroid gland as previously described [14]. Briefly, under anesthesia the trachea was exposed and under a stereoscope microscope the parathyroid glands were dissected from the thyroid gland and reimplanted into the surrounding neck muscles. The thyroid gland was then carefully dissected, to avoid injury to the laryngeal nerves, and completely excised. The effectiveness of this procedure has been demonstrated previously [15]. Blood levels of T3 diminished from 0.949 0.12 to 0.0619 0.010 mg/dl. Hyperthyroidism was induced to male Wistar rats by a daily i.p. injection
of 2 mg/kg of 3,5,3%-triiodothyronine (T3) for 4 days. The blood levels of T3 were increased from 0.0949 0.12 to 5.249 2.04 mg/100 ml. T3 values were established by analyzing the plasma of control, hypothyroid and injected rats by radioimmunoassay. Heart damage by ischemia/reperfusion in control, hypothyroid and hyperthyroid rats was carried out as follows: the rats were anesthetized with sodium pentobarbital (55 mg/kg, i.p.), and through a tracheotomy were maintained under assisted respiration. One lead-II surface electrocardiograph was used to monitor heart rate; blood pressure was measured with a pressure transducer joined to a femoral cannula. The chest was opened by thoracotomy; the left coronary artery was isolated near its origin by an intramural 6-0 silk loop. The occlusion of the artery was performed by passing a short tube over the vessel and clamping it firmly. The ischemic period lasted 5 min. This time was selected in agreement with previous reports [8,16,17]. The ischemic area was evaluated by autoradiography, after the injection of 166 mCi of 201Tl through a cannula inserted in the femoral artery of the right leg. Samples of the labeled-tissue from left ventricle free wall were placed in a cassette for exposing to X-ray film for 16 h. Reperfusion was started by removing the clamps. Those heart beats that were able to rise blood pressure were considered normal. At the end of the experiment, 1.5 ml of blood was withdrawn from the left ventricular cavity to determine the enzyme activity of CK, and ASAT, as reported [18,19]. Also, at the end of the experiment, samples of the left ventricular free wall were obtained for histological studies. The tissue was fixed in 10% formol-buffer and sliced at 4 mm thickness, Masson dye was used to visualize the fibers. Student’s t-test for paired samples was used to compare the data of heart rate and serum levels of the enzymes from hypothyroid, hyperthyroid and control rats. Values are expressed as means 9 S.E.M., significance was set at P B0.05.
3. Results Fig. 1 shows ECG tracings from control, hyperthyroid and hypothyroid rats. Panel A depicts the
I. Bobadilla et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 499–506
electric profile of hearts from control rats. As observed, during the occlusion period the heart was in a sinus rhythm; nevertheless, following removal of the occlusion, reperfusion arrhythmias were evident and they proceeded until the end of the experiment, i.e. 10 min later. Panel B shows ECG tracings from hyperthyroid rats, it is clearly
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observed that during the ischemia period there was an increase in the cardiac frequency, compared with that observed in control hearts. When blood-reflow was established, the incidence of ventricular tachycardia was larger when compared with euthyroid rat hearts. Interestingly, Panel C shows that hypothyroidism provides protection
Fig. 1. Electrocardiogram and blood pressure tracings of control (A), hyperthyroid (B), and hypothyroid (C) rats. The tracing represents one of 20 separate experiments.
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I. Bobadilla et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 499–506
Fig. 2. Autoradiography of the 201Thalium labeled myocardium from ischemic and non-ischemic hearts from hypothyroid rats. Experimental conditions as were described in Section 2. The image represents one of eight separate experiments. In (A) and (B) are shown tissue samples from ventricle non-subjected and subjected to ischemia, repectively. In (C) and (D) are shown autoradiography images from non-ischemic and ischemic myocardium, respectively.
against reperfusion-induced damage. As observed, abnormal beats were absent and the hearts remained in sinus rhythm through the length of the experiment. It should be noted that blood pressure, lower traces in their respective panels, was almost abolished in control and hyperthyroid rats. In contrast, in hypothyroid rats the magnitude of this variable was maintained within normal values. The experiment shown in Fig. 2 was performed to assess the magnitude of the ischemic area in samples of tissue from the free wall of the left ventricle from hypothyroid rats. As illustrated, the autoradiography shows that non-ischemic hearts the radionuclide was uniformly distributed (image A). Conversely, a non-radiolabeled area was present in ischemic heart (image B). Images C and D were obtained with samples from control and hyperthyroid rats. An objective analysis of cardiac frequency (CF), ventricular tachycardia (VT), ventricular
fibrillation (VF), and normal beats (NB) during reperfusion, in hearts from control, hyperthyroid and hypothyroid rats is shown in Table 1. As indicated, cardiac frequency before and during ischemia was higher in control and hyperthyroid than in hypothyroid rats. Ventricular tachycardia in control and hyperthyroid rats had a similar duration period, that is, around 11 s/min. In contrast, VT was almost absent in hypothyroid rats. Table 1 also shows the analysis of the duration of premature ventricular contractions (PVC). Although in control and hyperthyroid rats the duration periods were a few seconds per minute, remarkably these abnormalities were not present in hypothyroid rats. A similar picture was observed when VF was analyzed, i.e. a complete absence in hypothyroid rats. Corresponding with the latter findings, when the incidence of normal beats was analyzed, a clear-cut difference in favor of hypothyroidism, against control, and hyperthyroidism was observed.
I. Bobadilla et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 499–506
Analysis of the serum levels of CK and ASAT has been frequently used to evaluate tissue injury [18,19]. Therefore, such analysis was carried out and the results are shown in Table 2. The values indicate that, after reperfusion, CK was increased in the serum of euthyroid and hyperthyroid rats by approximately sixfold. From the basal values of 54.74929.85 IU/l in euthyroid untreated rats, to 338.989122.75 and 325.719144.84 IU/l, in reperfused euthyroid and hyperthyroid rats respectively. In comparison, in hypothyroid rats the serum values remained unchanged, i.e. 68.29 49.22 IU/l. Regarding the serum level of ASAT it was found that this enzyme was three times higher in reperfused hyperthyroid rats (29.29 11.03 IU/ l), than in reperfused euthyroid rats (9.949 1.78 IU/l). The values found in hypothyroid rats were similar (149 3.08 IU/l) to those observed in euthyroid untreated rats. These results confer further support to the notion that hypothyroidism renders resistance to reperfusion-induced heart damage. Fig. 3A shows light microscopy of myocardial tissue from untreated euthyroid rats. A characteristic image of a normal tissue is shown, i.e. ab-
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sence of edema and of contraction bands. In contrast, in accordance with reported data [8,17,20], light microscopy in the myocardial tissue of control reperfused rats shows the development of edema, which causes separation of the myocardial fibers. The formation of contraction bands is also shown (Fig. 3B). Similar abnormalities were found in myocardial tissue of hyperthyroid rats (Fig. 3C). However, as seen in Fig. 3D, myocardial fibers from hypothyroid rats retained their striae although some degree of edema is observed.
4. Discussion Thyroid hormone excess causes heart disease frequently manifested by atrial and ventricular extra-systoles, atrial fibrillation and ventricular repolarization abnormalities [21]. In turn, hypothyroidism promotes diastolic dysfunction, i.e. significant prolongation of the isovolumic relaxation time, and an increased A wave [22]. In this work we demonstrate that hyperthyroidism sensitizes rat heart to reperfusion injury, characterized
Table 1 Analysis of the different types of arrhythmias produced under reperfusiona
CF before ischemia (s/min) CF during ischemia (s/min) VT (s/min) PVC (s/min) VF (s/min) NB (s/min)
Control
Hyperthyroid
Hypothyroid
324.35 937.23 295.02942.06 11.7595.62 2.2292.33 4.739 5.60 1.1491.85
454.65 9 56.48 413.03 976.62 11.22 96.91 0.9 92.59 5.94 97.42 1.04 92.33
195.25 933.01 185.86 9 25.11 0.4 91.78 0 0 19.5 9 2.23
a Experimental conditions as were described for Fig. 1. Where indicated: CF, cardiac frequency; VF, ventricular fibrillation; VT, ventricular tachycardia; PVC, premature ventricular contractions; NB, normal beats. The values are expressed as mean 9S.E.M. of 20 separate experiments.
Table 2 Plasma concentrations of creatine kinase (CK) and aspartate amino transferase (ASAT) enzymesa Enzyme
Untreated
Control
Hyperthyroid
Hypothyroid
CK (IU/L) ASAT (IU/L)
54.749 29.85 9.949 1.78
338.98 9122.75 15.30 9 8.40
325.71 9 144.84 29.20 9 11.03
68.2 9 49.22 14 9 3.08
a Untreated rats were those not submitted to ischemia/reperfusion. The analysis were carried out as described in Section 2. The values represent the average 9 S.E.M. of 20 separate experiments.
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Fig. 3. Histological image of cardiac tissue after reperfusion. Histological sections from heart tissue of control rats (A) show heart image from untreated euthyroid rats, as observed there is absence of edema and contraction bands. In (B) is shown the image of a reperfused heart from euthyroid rats. The formation of contraction bands are shown. In (C) is indicated that hearts from hyperthyroid rats present an image of contraction bands similar to that observed in euthyroid rat hearts. In (D) is illustrated an image of a transversal cut in the ventricular free wall from hypothyroid rats. It is observed that although some edema is present, myocardial fibers retain their striae. The picture (400 × ) is representative of samples of ten separate experiments.
by severe arrhythmias, tissue lesion, and enzyme release. Remarkably, it is demonstrated that hypothyroidism renders resistance to reperfusion-induced damage. There is a widespread knowledge that mitochondrial permeability transition underlies the pathogenesis of myocardial reperfusion injury [8,11,12,23,24]. The non-specific mitochondrial permeability may be induced by reactive oxygen
species and Ca2 + overload [9,12]. In this regard, Ferna´ ndez and Videla [25] demonstrated that hyperthyroidism increases the rate of superoxide radical generation in liver submitochondrial particles. In addition it has been demonstrated that in rat liver mitochondria L-thyroxin stimulates Ca2 + -induced membrane leakage [26–28]. The above must be taken into account to explain the increased susceptibility of hyperthyroid rats to my-
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ocardial damage by reperfusion. Accordingly, protection of mitochondria against oxidative stress, or Ca2 + -dependent permeabilization resulted in protection against reperfusion-induced damage. Crompton et al. [29], and Arteaga et al. [8] have shown that the immunosuppressive drug cyclosporin A, which blocks the mitochondrial non-specific pore [30], prevents reperfusion-induced myocardial damage. By the same token, Cha´ vez et al. [31], and Carbajal et al. [32], have shown that the analgesic Ketorolac, that induces mitochondrial Ca2 + release [33], protects from reperfusion myocardial injury. Considering the above, the lack of reperfusion damage in hearts from hypothyroid animals must be ascribed to the finding that mitochondria from hypothyroid rats are resistant to Ca2 + -induced permeability transition [13]. It appears that the cause of the resistance could be due to the different composition of the lipid milieu of cell membranes [34,35]. In hypothyroidism, the inner mitochondrial membrane contains a low concentration of cardiolipin since cardiolipin synthesis depends on a mitochondrial enzyme, which is stimulated by T3 [36]. Cardiolipin is a phospholipid required for the full activity of the adenine nucleotide translocase [37], which under special circumstances is converted to the non-specific pore [9]. Therefore, a lack of cardiolipin would restrain pore formation, preserving the intactness of mitochondria, and in turn provide protection against reperfusion injury. In conclusion, our results show that hypothyroidism renders myocardial tissue resistant to reperfusion injury. The lack of electrocardiographic abnormalities, release of marker enzymes and the preservation of the anatomy of the myocardium demonstrated this. Finally, the data obtained from this work would be useful to evaluate those patients, candidates to be subjected to reperfusion, considering that hypothyroidism would confer on them less risk than that of hyperthyroidism.
Acknowledgements This work was partially supported by the Grant 28666N from CONACyT.
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[17] J.F. Te´ llez, K. Carbajal, A. Ca´ rabez, E. Cha´ vez, Effect of perezone on arrhythmias and markers of cell injury during reperfusion in the anesthetized rat, Life Sci. 65 (1999) 1615 – 1623. [18] M.L. Entman, L. Michel, R.D. Rosee, W.J. Dreyed, D.C. Anderson, A.A. Taylor, W. Smith, Inflammation in the course of early myocardial ischemia, FASEB J. 5 (1991) 2529 – 2537. [19] A. Karmen, Transaminase activity in human blood, J. Clin. Invest. 11 (1955) 126 –133. [20] M. Ashraf, H.D. Sybers, Scanning electron microscopy of the heart after coronary occlusion, Lab. Invest. 32 (1975) 157 – 162. [21] J.C. Forfar, G.C. Caldwell, Hyperthyroid heart disease, Clin. Endocrinol. Methods 14 (1985) 491 – 508. [22] M. Gonberg-Maitland, W.H. Frishman, Thyroid hormone and cardiovascular disease, Am. Heart. J. 135 (1998) 187 – 196. [23] M. Crompton, A. Costi, L. Hayat, Evidence for the presence of a reversible Ca2 + -dependent pore activated by oxidative stress in heart mitochondria, Biochem. J. 245 (1987) 915 –918. [24] M. Crompton, A. Costi, A heart mitochondrial Ca2 + -dependent pore of possible relevance to re-perfusion-induced injury. Evidence that ADP facilitates pore interconversion between the closed and open states, Biochem. J. 266 (1990) 33 –39. [25] V. Ferna´ ndez, L.A. Videla, Influence of hyperthyroidism on superoxide radical and hydrogen peroxide production by rat liver submitochondrial particles, Free Rad. Res. 18 (1993) 329 – 335. [26] V. Kalderon, O. Hermesh, J. Bar-Tana, Mitochondrial permeability transition is induced by in vivo thyroid hormone treatment, Endocrinology 136 (1995) 3552 – 3556. [27] N.V. Malkevitch, V.I. Dedukhova, R.A. Simonian, V.P. Skulachev, A.A. Starkov, Thyroxin induces cyclosporin A-insensitive Ca2 + -dependent reversible permeability transition pore in rat liver mitochondria, FEBS Lett. 412
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Hypothyroidism provides resistance to reperfusion injury following myocardium ischemia Israel Bobadilla a, Martha Franco b, David Cruz c, Jose` Zamora d, Sandra G. Robles a, Edmundo Cha´vez a,* Departamento de Bioquı´mica, Instituto Nacional de Cardiologı´a, Ignacio Cha´6ez, Juan Badiano c 1, Mexico, D.F. 014080, Mexico b Departamento de Nefrologı´a, Instituto Nacional de Cardiologı´a, Ignacio Cha´6ez, Mexico, D.F. 014080, Mexico c Departamento de Patologı´a, Instituto Nacional de Cardiologı´a, Ignacio Cha´6ez, Mexico, D.F. 014080, Mexico d Departamento de Endocrinologı`a, Instituto Nacional de Cardiologı´a, Ignacio Cha´6ez, Mexico, D.F. 014080, Mexico a
Received 16 May 2000; accepted 17 January 2001
Abstract A growing body of evidence has demonstrated that reperfusion injury may be mediated, in part, by mitochondrial Ca2 + overload that promotes non-selective permeability of the inner membrane. In this regard it is known that mitochondria from hypothyroid rats are resistant to membrane damage as induced by Ca2 + . The purpose of this study was to evaluate the sensitivity of hearts from hypothyroid rats, to the damage by reperfusion, after an ischemic period of 5 min. The results were compared with those from control and hyperthyroid rats. Hypothyroidism was established by surgical removal of the thyroid gland; in turn hyperthyroidism was induced after a daily injection of 2 mg/kg of 3,5,3%-triiodothyronine for 4 days. ECG tracings from hypothyroid rats showed a total absence of post-reperfusion arrhythmias conversely to what was observed in control and hyperthyroid rats. The release of creatine kinase and aspartate amino transferase to the plasma in hypothyroid rats was found to be lower than that found in hyperthyroid and euthyroid rats. The histological studies showed that myocardial fibers from hypothyroid rats were in good condition and retained their striae and a remarkable near absence of edema was clearly observed. © 2001 Published by Elsevier Science Ltd. Keywords: Hypothyroidism; Ischemia/reperfusion; Rat heart; Myocardium reperfusion; Reperfusion injury
1. Introduction Ischemia, when sufficiently prolonged, produces non-reversible tissue destruction. In the context of myocardial ischemia, advances in clinical and sur* Corresponding author. Tel.: + 52-5-5732911; fax: + 52-55730926. E-mail address: [email protected] (E. Cha´vez).
gical practice have allowed reperfusion of viable heart tissue, i.e. cardioplegic solutions, implant of intracoronary stents, or coronary by-pass. However, abrupt reoxygenation causes further substantial cell injury produced by oxygen-derived reactive species formed through dissimilar mechanisms [1–4]. In addition, the resulting cellular Ca2 + overload contributes significantly to the myocardial insult [5–7]. Regarding the latter, in a
1357-2725/01/$ - see front matter © 2001 Published by Elsevier Science Ltd. PII: S 1 3 5 7 - 2 7 2 5 ( 0 1 ) 0 0 0 1 6 - 4
500
I. Bobadilla et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 499–506
previous work we demonstrated that the immunosuppressive drug cyclosporin A (CSA) avoids heart damage promoted by ischemia-reperfusion [8]. The ability of CSA to prevent mitochondrial membrane leakage, caused by Ca2 + overload is well documented [9– 11]. At present, there is a consensus about the central role of mitochondrial permeability transition in heart reperfusion injury [11,12]. Recently we found that hypothyroidism renders liver mitochondria resistant to permeability transition [13]. We proposed that the lack of cardiolipin in the lipid phase of membrane restrains the shift of adenine nucleotide translocase to a transmembrane pore. Taking together these previous published data we decided to explore the possibility that heart tissue of hypothyroid rats might be less sensitive to ischemia-reperfusion injury. The results indicate that, certainly, hypothyroidism conferred protection against reperfusion arrhythmias, and the release of creatine kinase (CK) and aspartate amino transferase (ASAT). Furthermore, histological studies show that myocardial tissue from hypothyroid rat hearts preserved its normal structure. When compared with experiments performed in hyperthyroid rats the picture was quite different. In these rats there was an increased incidence of ventricular tachycardia, an increase in the serum levels of CK and ASAT, as well as a generalized injury in the myocardial structure.
2. Materials and methods Hypothyroidism was induced in male Wistar rats, weighing between 250 and 300 g, by removing the thyroid gland as previously described [14]. Briefly, under anesthesia the trachea was exposed and under a stereoscope microscope the parathyroid glands were dissected from the thyroid gland and reimplanted into the surrounding neck muscles. The thyroid gland was then carefully dissected, to avoid injury to the laryngeal nerves, and completely excised. The effectiveness of this procedure has been demonstrated previously [15]. Blood levels of T3 diminished from 0.949 0.12 to 0.0619 0.010 mg/dl. Hyperthyroidism was induced to male Wistar rats by a daily i.p. injection
of 2 mg/kg of 3,5,3%-triiodothyronine (T3) for 4 days. The blood levels of T3 were increased from 0.0949 0.12 to 5.249 2.04 mg/100 ml. T3 values were established by analyzing the plasma of control, hypothyroid and injected rats by radioimmunoassay. Heart damage by ischemia/reperfusion in control, hypothyroid and hyperthyroid rats was carried out as follows: the rats were anesthetized with sodium pentobarbital (55 mg/kg, i.p.), and through a tracheotomy were maintained under assisted respiration. One lead-II surface electrocardiograph was used to monitor heart rate; blood pressure was measured with a pressure transducer joined to a femoral cannula. The chest was opened by thoracotomy; the left coronary artery was isolated near its origin by an intramural 6-0 silk loop. The occlusion of the artery was performed by passing a short tube over the vessel and clamping it firmly. The ischemic period lasted 5 min. This time was selected in agreement with previous reports [8,16,17]. The ischemic area was evaluated by autoradiography, after the injection of 166 mCi of 201Tl through a cannula inserted in the femoral artery of the right leg. Samples of the labeled-tissue from left ventricle free wall were placed in a cassette for exposing to X-ray film for 16 h. Reperfusion was started by removing the clamps. Those heart beats that were able to rise blood pressure were considered normal. At the end of the experiment, 1.5 ml of blood was withdrawn from the left ventricular cavity to determine the enzyme activity of CK, and ASAT, as reported [18,19]. Also, at the end of the experiment, samples of the left ventricular free wall were obtained for histological studies. The tissue was fixed in 10% formol-buffer and sliced at 4 mm thickness, Masson dye was used to visualize the fibers. Student’s t-test for paired samples was used to compare the data of heart rate and serum levels of the enzymes from hypothyroid, hyperthyroid and control rats. Values are expressed as means 9 S.E.M., significance was set at P B0.05.
3. Results Fig. 1 shows ECG tracings from control, hyperthyroid and hypothyroid rats. Panel A depicts the
I. Bobadilla et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 499–506
electric profile of hearts from control rats. As observed, during the occlusion period the heart was in a sinus rhythm; nevertheless, following removal of the occlusion, reperfusion arrhythmias were evident and they proceeded until the end of the experiment, i.e. 10 min later. Panel B shows ECG tracings from hyperthyroid rats, it is clearly
501
observed that during the ischemia period there was an increase in the cardiac frequency, compared with that observed in control hearts. When blood-reflow was established, the incidence of ventricular tachycardia was larger when compared with euthyroid rat hearts. Interestingly, Panel C shows that hypothyroidism provides protection
Fig. 1. Electrocardiogram and blood pressure tracings of control (A), hyperthyroid (B), and hypothyroid (C) rats. The tracing represents one of 20 separate experiments.
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Fig. 2. Autoradiography of the 201Thalium labeled myocardium from ischemic and non-ischemic hearts from hypothyroid rats. Experimental conditions as were described in Section 2. The image represents one of eight separate experiments. In (A) and (B) are shown tissue samples from ventricle non-subjected and subjected to ischemia, repectively. In (C) and (D) are shown autoradiography images from non-ischemic and ischemic myocardium, respectively.
against reperfusion-induced damage. As observed, abnormal beats were absent and the hearts remained in sinus rhythm through the length of the experiment. It should be noted that blood pressure, lower traces in their respective panels, was almost abolished in control and hyperthyroid rats. In contrast, in hypothyroid rats the magnitude of this variable was maintained within normal values. The experiment shown in Fig. 2 was performed to assess the magnitude of the ischemic area in samples of tissue from the free wall of the left ventricle from hypothyroid rats. As illustrated, the autoradiography shows that non-ischemic hearts the radionuclide was uniformly distributed (image A). Conversely, a non-radiolabeled area was present in ischemic heart (image B). Images C and D were obtained with samples from control and hyperthyroid rats. An objective analysis of cardiac frequency (CF), ventricular tachycardia (VT), ventricular
fibrillation (VF), and normal beats (NB) during reperfusion, in hearts from control, hyperthyroid and hypothyroid rats is shown in Table 1. As indicated, cardiac frequency before and during ischemia was higher in control and hyperthyroid than in hypothyroid rats. Ventricular tachycardia in control and hyperthyroid rats had a similar duration period, that is, around 11 s/min. In contrast, VT was almost absent in hypothyroid rats. Table 1 also shows the analysis of the duration of premature ventricular contractions (PVC). Although in control and hyperthyroid rats the duration periods were a few seconds per minute, remarkably these abnormalities were not present in hypothyroid rats. A similar picture was observed when VF was analyzed, i.e. a complete absence in hypothyroid rats. Corresponding with the latter findings, when the incidence of normal beats was analyzed, a clear-cut difference in favor of hypothyroidism, against control, and hyperthyroidism was observed.
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Analysis of the serum levels of CK and ASAT has been frequently used to evaluate tissue injury [18,19]. Therefore, such analysis was carried out and the results are shown in Table 2. The values indicate that, after reperfusion, CK was increased in the serum of euthyroid and hyperthyroid rats by approximately sixfold. From the basal values of 54.74929.85 IU/l in euthyroid untreated rats, to 338.989122.75 and 325.719144.84 IU/l, in reperfused euthyroid and hyperthyroid rats respectively. In comparison, in hypothyroid rats the serum values remained unchanged, i.e. 68.29 49.22 IU/l. Regarding the serum level of ASAT it was found that this enzyme was three times higher in reperfused hyperthyroid rats (29.29 11.03 IU/ l), than in reperfused euthyroid rats (9.949 1.78 IU/l). The values found in hypothyroid rats were similar (149 3.08 IU/l) to those observed in euthyroid untreated rats. These results confer further support to the notion that hypothyroidism renders resistance to reperfusion-induced heart damage. Fig. 3A shows light microscopy of myocardial tissue from untreated euthyroid rats. A characteristic image of a normal tissue is shown, i.e. ab-
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sence of edema and of contraction bands. In contrast, in accordance with reported data [8,17,20], light microscopy in the myocardial tissue of control reperfused rats shows the development of edema, which causes separation of the myocardial fibers. The formation of contraction bands is also shown (Fig. 3B). Similar abnormalities were found in myocardial tissue of hyperthyroid rats (Fig. 3C). However, as seen in Fig. 3D, myocardial fibers from hypothyroid rats retained their striae although some degree of edema is observed.
4. Discussion Thyroid hormone excess causes heart disease frequently manifested by atrial and ventricular extra-systoles, atrial fibrillation and ventricular repolarization abnormalities [21]. In turn, hypothyroidism promotes diastolic dysfunction, i.e. significant prolongation of the isovolumic relaxation time, and an increased A wave [22]. In this work we demonstrate that hyperthyroidism sensitizes rat heart to reperfusion injury, characterized
Table 1 Analysis of the different types of arrhythmias produced under reperfusiona
CF before ischemia (s/min) CF during ischemia (s/min) VT (s/min) PVC (s/min) VF (s/min) NB (s/min)
Control
Hyperthyroid
Hypothyroid
324.35 937.23 295.02942.06 11.7595.62 2.2292.33 4.739 5.60 1.1491.85
454.65 9 56.48 413.03 976.62 11.22 96.91 0.9 92.59 5.94 97.42 1.04 92.33
195.25 933.01 185.86 9 25.11 0.4 91.78 0 0 19.5 9 2.23
a Experimental conditions as were described for Fig. 1. Where indicated: CF, cardiac frequency; VF, ventricular fibrillation; VT, ventricular tachycardia; PVC, premature ventricular contractions; NB, normal beats. The values are expressed as mean 9S.E.M. of 20 separate experiments.
Table 2 Plasma concentrations of creatine kinase (CK) and aspartate amino transferase (ASAT) enzymesa Enzyme
Untreated
Control
Hyperthyroid
Hypothyroid
CK (IU/L) ASAT (IU/L)
54.749 29.85 9.949 1.78
338.98 9122.75 15.30 9 8.40
325.71 9 144.84 29.20 9 11.03
68.2 9 49.22 14 9 3.08
a Untreated rats were those not submitted to ischemia/reperfusion. The analysis were carried out as described in Section 2. The values represent the average 9 S.E.M. of 20 separate experiments.
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Fig. 3. Histological image of cardiac tissue after reperfusion. Histological sections from heart tissue of control rats (A) show heart image from untreated euthyroid rats, as observed there is absence of edema and contraction bands. In (B) is shown the image of a reperfused heart from euthyroid rats. The formation of contraction bands are shown. In (C) is indicated that hearts from hyperthyroid rats present an image of contraction bands similar to that observed in euthyroid rat hearts. In (D) is illustrated an image of a transversal cut in the ventricular free wall from hypothyroid rats. It is observed that although some edema is present, myocardial fibers retain their striae. The picture (400 × ) is representative of samples of ten separate experiments.
by severe arrhythmias, tissue lesion, and enzyme release. Remarkably, it is demonstrated that hypothyroidism renders resistance to reperfusion-induced damage. There is a widespread knowledge that mitochondrial permeability transition underlies the pathogenesis of myocardial reperfusion injury [8,11,12,23,24]. The non-specific mitochondrial permeability may be induced by reactive oxygen
species and Ca2 + overload [9,12]. In this regard, Ferna´ ndez and Videla [25] demonstrated that hyperthyroidism increases the rate of superoxide radical generation in liver submitochondrial particles. In addition it has been demonstrated that in rat liver mitochondria L-thyroxin stimulates Ca2 + -induced membrane leakage [26–28]. The above must be taken into account to explain the increased susceptibility of hyperthyroid rats to my-
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ocardial damage by reperfusion. Accordingly, protection of mitochondria against oxidative stress, or Ca2 + -dependent permeabilization resulted in protection against reperfusion-induced damage. Crompton et al. [29], and Arteaga et al. [8] have shown that the immunosuppressive drug cyclosporin A, which blocks the mitochondrial non-specific pore [30], prevents reperfusion-induced myocardial damage. By the same token, Cha´ vez et al. [31], and Carbajal et al. [32], have shown that the analgesic Ketorolac, that induces mitochondrial Ca2 + release [33], protects from reperfusion myocardial injury. Considering the above, the lack of reperfusion damage in hearts from hypothyroid animals must be ascribed to the finding that mitochondria from hypothyroid rats are resistant to Ca2 + -induced permeability transition [13]. It appears that the cause of the resistance could be due to the different composition of the lipid milieu of cell membranes [34,35]. In hypothyroidism, the inner mitochondrial membrane contains a low concentration of cardiolipin since cardiolipin synthesis depends on a mitochondrial enzyme, which is stimulated by T3 [36]. Cardiolipin is a phospholipid required for the full activity of the adenine nucleotide translocase [37], which under special circumstances is converted to the non-specific pore [9]. Therefore, a lack of cardiolipin would restrain pore formation, preserving the intactness of mitochondria, and in turn provide protection against reperfusion injury. In conclusion, our results show that hypothyroidism renders myocardial tissue resistant to reperfusion injury. The lack of electrocardiographic abnormalities, release of marker enzymes and the preservation of the anatomy of the myocardium demonstrated this. Finally, the data obtained from this work would be useful to evaluate those patients, candidates to be subjected to reperfusion, considering that hypothyroidism would confer on them less risk than that of hyperthyroidism.
Acknowledgements This work was partially supported by the Grant 28666N from CONACyT.
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